Backflow prevention for high pressure gradient systems

ABSTRACT

Gradient performance with high pressure gradient solvent delivery system is optimized by approximation of infinite stroke volume of high pressure pumps by the addition of pulse dampening with backflow prevention to each high pressure pump. The backflow prevention adds sufficient minimum flow resistance, thereby enhancing the performance of the pulse dampening over a wider range of flow rates resulting in consistent gradient performance.

FIELD OF THE INVENTION

[0001] The present invention relates to liquid chromatographyinstrumentation and solvent delivery systems, and more particularly to amethod and apparatus for control of chromatographic pumping systems.

BACKGROUND OF THE INVENTION

[0002] High-pressure liquid chromatography (HPLC) solvent deliverysystems are used to source single-component liquids or mixtures ofliquids (both known as “mobile phase”) at pressures which can range fromsubstantially atmospheric pressure to pressures on the order of tenthousand pounds per square inch. These pressures are required to forcethe mobile phase through the fluid passageways of a stationary phasesupport, where separation of dissolved analytes can occur. Thestationary phase support may comprise a packed bed of particles, amembrane or collection of membranes, a porous monolithic bed, or an opentube. Often, analytical conditions require the mobile phase compositionto change over the course of the analysis (this mode being termed“gradient elution”). In gradient elution, the viscosity of the mobilephase may change and the pressure necessary to maintain the requiredvolumetric flow rate will change accordingly.

[0003] In liquid chromatography, the choice of an appropriate separationstrategy (including hardware, software, and chemistry) results in theseparation of an injected sample mixture into its components, whichelute from the column in reasonably distinct zones or “bands”. As thesebands pass through a detector, their presence can be monitored and adetector output (usually in the form of an electrical signal) can beproduced. The pattern of analyte concentration within the eluting bands,which can be represented by means of a time-varying electrical signal,gives rise to the nomenclature of a “chromatographic peak”. Peaks may becharacterized with respect to their “retention time”, which is the timeat which the center of the band transits the detector, relative to thetime of injection (i.e. time-of-injection is equal to zero). In manyapplications, the retention time of a peak is used to infer the identityof the eluting analyte, based upon related analyses with standards andcalibrants. The retention time for a peak is strongly influenced by themobile phase composition, and by the accumulated volume of mobile phasewhich has passed over the stationary phase.

[0004] The utility of chromatography relies heavily on run-to-runreproducibility, such that standards or calibrants can be analyzed inone set of runs, followed by test samples or unknowns, followed by morestandards, in order that confidence can be had in the resulting data.Known pumping systems exhibit some non-ideal characteristics whichresult in diminished separation performance and diminished run-to-runreproducibility. Among the non-ideal pump characteristics exhibited inknown pumping systems are, generally, fluctuations in solventcomposition and/or fluctuations in volumetric flow rate.

[0005] Volumetric flow fluctuations present in known HPLC pumpingsystems disadvantageously cause retention time(s) to vary for a givenanalyte. That is, the amount of time that an analyte is retained in thestationary phase fluctuates undesirably as a function of the undesirablevolumetric flow fluctuations. This creates difficulties in inferring theidentity of a sample from the retention behavior of the components.Volumetric flow fluctuations from individual pumps can result influctuations in solvent composition when the output of multiple pumps issummed to provide a solvent composition.

[0006] Fluctuations in solvent composition present in known HPLC pumpingsystems can disadvantageously result in interactions with the system'sanalyte detector and produce perturbations which are detected as if theyarose from the presence of a sample. In effect, an interference signalis generated. This interference signal is summed with the actual signalattributable to the analyte, producing errors when the quantity of anunknown sample is calculated from the area of the eluting sample peak.

[0007] The prior art is replete with techniques and instrumentimplementations aimed at controlling solvent delivery and minimizingperturbations in the output of delivery systems for analyticalinstrumentation. Myriad pump configurations are known which deliverfluid at high pressure for use in applications such as liquidchromatography. Known pumps, such as one disclosed in U.S. Pat. No.4,883,409 (“the '409 patent”) incorporate at least one plunger or pistonwhich is reciprocated within a pump chamber into which fluid isintroduced. A controlled reciprocation frequency and stroke length ofthe plunger within the pump chamber determines the flow rate of fluidoutput from the pump. However, the assembly for driving the plunger isan elaborate combination of elements that can introduce undesirablemotion in the plunger as it is driven, which motion makes it difficultto precisely control the solvent delivery system output and results inwhat is termed “noise” or detectable perturbations in a chromatographicbaseline. Much of this noise does not result from random statisticalvariation in the system, rather much of it is a function of a mechanical“signature” of the pump. Mechanical signature is correlated tomechanically related phenomena such as anomalies in ball and screwdrives, gears, and/or other components used in the pump to effect thelinear motion that drives the piston(s), or it is related to higherlevel processes or physical phenomena such as the onset or completion ofsolvent compression, or the onset of solvent delivery from the pumpchamber.

[0008] Typical systems known for delivery of liquids in liquidchromatography applications, such as disclosed in the '409 patent andfurther in U.S. Pat. No. 5,393,434, implement dual piston pumps havingtwo interconnected pump heads each with a reciprocating plunger. Theplungers are driven with a predetermined phase difference in order tominimize output flow variations. Piston stroke length and strokefrequency can be independently adjusted when the pistons areindependently, synchronously driven. Precompression can be effected ineach pump cylinder in any given pump cycle to compensate for varyingfluid compressibilities in an effort to maintain a substantiallyconstant system pressure and output flow rate.

[0009] There are two widely used means to create gradient HPLC pumps.The solvents can be blended on the intake side of the pump. This isknown in the art as low pressure gradient mixing. The alternative is theuse of so-called high pressure gradient systems in which each individualsolvent is delivered by a separate pump.

[0010] The fundamental scalar of all forms of gradient chromatography isthe void volume of the separation column. The void volume of an HPLCcolumn is the sum of the inter and intra particle volumes of the columnthat are filled with mobile phase. The void volume is the minimum volumerequired to elute an unretained solute. The gradient delay volume is thevolume of the mobile phase delivered from the time the gradient isinitiated to when the change in composition first arrives at the column.The delay volume is the volumetric overhead of the gradient solventdelivery system; it adds to the time required to complete the separationand to prepare the column for the next injection. The delay volumeshould be minimized and ideally should be no more than two times largerthan the void volume of the column.

[0011] When two or more high pressure pumps are combined to form agradient solvent delivery system, their outputs are combined with theresulting possibility that there can be fluidic cross talk between thehigh pressure pumps during their individual piston crossovers. One priorapproach to avoid fluidic cross talk has been the use of pulse dampenerswithin the gradient solvent delivery system as shown in FIG. 1.

[0012] When individual pulse dampeners are placed up-stream from whereoutput of the solvents meet and the total flow is small relative to thevolume of the pulse dampeners, there will be significant crosstalkbetween the pumps since the two pumps are not synchronous in theirrespective piston crossovers. This crosstalk occurs because the fluidcontained in the off line pulse dampener can be compressed making it thelow impedance path for the on-line pump. As such, this up-streamplacement of the pulse dampeners results in a compromised flow rate andcomposition. The result of this fluidic crosstalk is shown in FIG. 2,which plots the delivery of a gradient marker from a solvent deliverysystem configured as shown in FIG. 1 at a low flow rate. As shown inFIG. 2, no gradient deliveries are identical and none correspond to theprogrammed gradient. This results in unsatisfactory and unpredictableseparations which cannot be reproduced.

[0013] In an alternative prior art approach, a capillary restrictor isused to generate back-pressure to energize the pulse dampeners. Acapillary of fixed length and internal diameter provides sufficientbackpressure to restrict, but unfortunately not prevent backflow, overnarrow ranges of flow rates.

[0014] A further approach to the use of pulse dampeners is to position apulse dampener downstream from the common mixing tee. While thisapproach is useful in gradient systems having large volumes, smallerscale volumes are problematic. The positioning of a pulse dampener afterthe common mixing tee greatly increases the delay volume within thegradient systems. Pulse dampeners are scaled to a specific and limitedflow rate range as they typically combine resistance to flow and acaptive capacitive volume of the mobile phase. The requirements ofeffective pulse dampening and minimizing delay volume will conflict asthe scale of the HPLC system with respect to column volume andvolumetric flow rate is reduced.

SUMMARY OF THE INVENTION

[0015] The present invention provides an improved method and apparatusfor improving the compositional accuracy of high pressure gradient pumpsfor HPLC by approximation of infinite stroke volume with backflowprevented pulse dampening. The backflow prevention, according to theinvention, adds sufficient minimum flow resistance thereby enhancing theperformance of the pulse dampening over a wider range of flow ratesresulting in consistent gradient performance.

[0016] According to the invention, pulse dampeners in conjunction withback flow preventors, which may be embodied as check valves or in-lineback-pressure regulators, ensure that the stored mobile phase iscompressed during the delivery cycle. When a backflow preventor with afixed minimum flow resistance is used, the effectiveness of the pulsedampener becomes substantially independent of the flow rate. The use ofbackflow preventors within the gradient system ensures that the storedmobile phase is compressed and has mechanical energy to return to thesystem at piston crossover.

[0017] The backflow preventors further ensure that the outlet checkvalves of the respective pump heads will experience sufficientbackpressure allowing for their proper functioning. This sufficientbackpressure is particularly helpful in systems having low flow rateswhen the backpressure generated by the column and tubing is limited.Additionally, backpressure allows the primary check valves of individualpumps to operate more consistently as the resulting backpressure ensuresproper seating of the outlet check valve on the pump head that is offline.

[0018] The proper placement of a backflow preventor according to theinvention reduces fluidic cross talk, optimizes the performance ofin-line pulse dampers and enhances the performance of the high pressurepumps as shown in FIG. 3., which plots the delivery of a gradient markerfrom a solvent delivery system configured with backflow preventionaccording to the invention. As depicted in FIG. 3, the gradientdeliveries are identical and correspond to the programmed gradient.

[0019] Advantageously, individual pumps deliver smooth flow by theaddition of suitable pulse dampeners with the further use of backflowpreventors that prevent fluidic cross talk between the two mobilephases. Because a pulse dampener is the fluidic equivalent of a low passfilter, when a small stroke volume is combined with a pulse dampener,the crossover perturbations occur at frequencies that are stronglyattenuated. The differences between individual pump heads areeffectively averaged by the use of pulse dampeners. Thus, the use ofsmall stoke volumes with efficient pulse dampening provide for uniformblending of solvents in high pressure gradient systems.

[0020] In an alternative illustrative embodiment a capillary restrictoris used to generate backpressure to energize the pulse dampener. Acapillary of fixed length and internal diameter provides sufficientbackpressure over a certain range of flow rates. The use of a capillaryrestrictor in series with a check valve can be used for systems havingconsistent flow rates.

[0021] In a further alternative illustrative embodiment the check valvesare incorporated into a mixing tee. This incorporation decreases thevolume of the mobile phase within the gradient system and thereforedecreases the delay volume of the gradient system.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The foregoing and other features and advantages of the presentinvention will be more fully understood from the following detaileddescription of illustrative embodiments, taken in conjunction with theaccompanying drawings in which:

[0023]FIG. 1 is a schematic of a standard high pressure gradient pump(prior art);

[0024]FIG. 2 demonstrates problems with fluidic crosstalk in highpressure gradient systems (prior art);

[0025]FIG. 3 illustrates the effect of pump crossover on solventcomposition without backflow prevention (prior art);

[0026]FIG. 4 demonstrates the use of pulse dampers with the addition ofin-line check valves according to the invention;

[0027]FIG. 5 illustrates the cumulative effect of pump crossover onsolvent composition (prior art);

[0028]FIG. 6 is a schematic of a high pressure gradient pumping systemaccording to the invention;

[0029]FIG. 7 is a schematic of a mixing tee having integrated checkvalves; and

[0030]FIG. 8 is a schematic of a mixing tee having integrated checkvalves and backpressure regulators.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present disclosure will be described in detail with respectto chromatographic applications with the understanding that embodimentsof the present invention are directed to industrial and process controlapplications as well.

[0032] As shown in FIG. 4 the effect of pump crossover on solventcomposition is illustrated.

[0033] Within this illustration, the total flow is 1 mL/min. A firstpump delivers ninety percent of the flow or approximately 900 μL/min. Asecond pump delivers ten percent of the flow or approximately 100μL/min. The stroke volume is approximately 100 μL for both pumps. Thereare nine crossovers of the first pump to one crossover of the secondpump to provide the desired composition. When the first pump crossesover there is a deficit in the first solvent of about 23 percent in flowrate and the composition is momentarily enriched in the second solvent.This deficit is illustrated by a first curve 301. A second curve 302shows the effect of reducing the magnitude of the flow rate deficit fromabout 23 percent loss of flow at crossover to 10 percent loss of flow bythe use of limited pulse dampening. The compositional perturbations or“noise” is reduced from about +3 percent of the second solvent deliveredto about +1 percent of the second solvent delivered. Further pulsedampening according to the invention would further reduce thecompositional noise.

[0034] The effect of this compositional noise on the retention times ofanalyte peaks is strongly dependent upon the degree of retention of theanalyte and is expressed in its k-prime (k′) value which is the numberof column volumes required to elute the analyte from the column. The k′value is computed from the following formula:

k′=(V _(r) −V _(o))/V _(o)  (Formula I)

[0035] where V_(r)=retention volume and V_(o)=column void volume.

[0036] When k′ is small, variations in mobile phase composition havelittle effect on retention volume, however when k′ is large smallvariations in mobile phase composition have a large effect on retentionvolume since k′ is exponentially proportional to the percent of thesecond solvent delivered.

[0037] The cumulative effect of pump crossovers on the percent of thesecond solvent delivered is illustrated in FIG. 5. The cumulative errorin the percent of the second solvent is strongly coupled to themagnitude of the gradient pulse. The resulting variance in retentiontimes will be strongly coupled with the degree of pulsation and themixing requirement ensuring a more uniform composition is directlycoupled to the instantaneous and the cumulative errors in the percent ofthe second solvent. When the pulsations are reduced according to theinvention the composition becomes inherently more uniform and requires asmaller volume to ensure its uniformity.

[0038] Turning to FIG. 6, an illustrative embodiment of the instantinvention is a high pressure gradient system in which each individualsolvent is delivered by a separate pump. This illustrative embodimenthas a first solvent delivery line 101 and a second solvent delivery line103. A first solvent is delivered to a first pump 105 within the firstsolvent delivery line 101 via a fluidic tee 104. The first pump 105 hasa first piston 107 and a second piston 109. In this illustrativeembodiment the first pump 105 is a Waters model HPLC pump 515, made byWaters Corporation of Milford Mass., which is a fluidic pump having afixed stroke length. It is contemplated within the scope of thisinvention that other pumps known in the art may be used.

[0039] The first solvent is delivered via the first pump 105 to a primevalve 111, such as Waters P/N WAT 207085, Waters Corporation, Milford,Mass, which also acts as a fluidic tee receiving the output from thefirst piston 107 and the second piston 109. The first solvent isdelivered to a first pulse dampener 112. The first pulse dampener 112,which in this illustrative embodiment is a Waters High Pressure Filter,P/N WAT207072, Waters Corporation, Milford, Mass., is a fluidic low passfilter that minimizes flow rate perturbation within the first solventdelivery line. It is contemplated within the scope of this inventionthat other pulse dampeners known in the art may be used.

[0040] The first solvent is pumped through the first pulse dampener 112and is delivered to a first backflow preventor 114. The first backflowpreventor 114, which in this illustrative embodiment is an UpchurchModel U-609, Upchurch Scientific, Oak Harbor, Wash., has a knownresistance to flow forces that causes a load onto the first pulsedampener 112 ensuring consistent operation of the first pulse dampener112. This resistance to flow can range from about 0 to 2,000 psi, and inthis first illustrative embodiment the resistance is approximately 250psi.

[0041] The first backflow preventor 114 is in fluid communication with acommon mixing tee 116 that directs the first solvent through a pressuretransducer 118 and into a vent valve 119, such as Rheodyne 7033,Rheodyne, LP., Rohnert Park, Calif. The vent valve 119 directs the firstsolvent to an injector and a chromatography column 120.

[0042] A second solvent is delivered to a second pump 122 within thesecond solvent delivery line 103 via a second fluidic tee 124. Thesecond pump 122 has a first piston 124 and a second piston 126. In thisillustrative embodiment the second pump 122 is a Waters model 515 HPLCpump, Waters Corporation Milford Mass., which is a fluidic pump having afixed stroke length. It is contemplated within the scope of thisinvention that other pumps known in the art may be used.

[0043] The second solvent is delivered via the second pump 122 to secondprime valve 128, such as Waters P/N WAT 207085, Waters Corporation,Milford, Mass., which also acts as a fluidic tee receiving the outputfrom the first piston 124 and the second piston 126. The second solventis delivered to a second pulse dampener 130. The second pulse dampener130 provides a fluidic low pass filter that minimizes flow rateperturbations within the second solvent delivery line. The secondsolvent is pumped to a second backflow preventor 132. The secondbackflow preventor 132 has a known resistance to flow forces that causesa pressure load onto the second pulse dampener 130 ensuring consistentoperation of the second pulse dampener 130. This resistance to flow canrange from 0 to 2000 psi, and in this first illustrative embodiment theresistance is approximately 250 psi.

[0044] The second backflow preventor 132 is in fluid communication withthe common mixing tee 116 that directs the first solvent through thepressure transducer 118 and into the vent valve 119 and the secondsolvent 102 through the pressure transducer 118 and into the vent valve119. The vent valve 119, such as a Rheodyne 7033, Rheodyne, LP., RohnertPark, Calif., directs the first solvent 101 and the second solvent 102to the chromatography column 120.

[0045] In an alternative embodiment of the invention the first backflowpreventor and the second backflow preventor are incorporated into thestructure of the mixing tee to minimize system volume. As illustrated inFIG. 7 the common mixing tee 201 has a first backflow preventor 203 anda second backflow preventor 205 incorporated into the structure of themixing tee 201. The mixing tee 201 has a first inlet port 207 in whichthe first backflow preventor 203 is incorporated, a second inlet port215 in which the second backflow preventor 205 is incorporated and anoutlet port 214 in which fluid flow from the first inlet port 207 andthe second inlet port 215 are directed.

[0046] The first backflow preventor 203 has a first ball bearing 211housed within a first check valve body 220. The first ball bearing 211is seated in a first check valve seat 213. The first ball bearing 211 isfabricated from materials that are inert to system solvents such assapphire and ceramic or the like. The first ball bearing 211 is encasedin a first check valve cartridge housing component 221 in a mannerallowing only forward fluid flow. The first check valve cartridgehousing component 221 is comprised of a top part 222 and a base part224, which forms the first check valve seat 213.

[0047] The second backflow preventor 205 has a second ball bearing 217housed within a second check valve body 223. The second ball bearing 217is seated in a second check valve seat 218. The second ball bearing 217is fabricated from materials that are inert to system solvents such assapphire and ceramic or the like. The second ball bearing 217 is encasedin a second check valve cartridge housing component 227 in a manner onlyallowing forward fluid flow. The second check valve cartridge housingcomponent 227 is comprised of a top part 229 and a base part 226 whichforms the second check valve seat 218.

[0048] In a further alternative embodiment of the invention the firstbackflow preventor and the second backflow preventor are incorporatedinto the structure of the mixing tee to minimize system volume. Asillustrated in FIG. 8 the common mixing tee 301 has a first backflowpreventor 303 and a second backflow preventor 305 incorporated into thestructure of the mixing tee 301. The mixing tee 301 has a first inletport 307 in which the first backflow preventor 303 is incorporated, asecond inlet port 315 in which the second backflow preventor 305 isincorporated and an outlet port 314 in which fluid flow from the firstinlet port 307 and the second inlet port 315 are directed.

[0049] The first backflow preventor 303 has a coil spring 309 thatapplies pressure to a first actuator 311. The first actuator 311 isseated into a first valve opening 313. The selected coil spring 309provides a certain resistance to flow by exerting pressure against thefirst actuator thereby sealing the first valve opening 313 until theresistance to flow is exceeded.

[0050] The second backflow preventor 305 has a coil spring 316 thatapplies pressure to a second actuator 317. The second actuator 317 isseated into a second valve opening 318. Again, the selected coil spring316 provides a certain resistance to flow by exerting pressure againstthe second actuator 317 thereby sealing the second valve opening 318until the resistance to flow is exceeded.

[0051] In a further alternative embodiment the pulse dampeners withinthe fluidic solvent delivery lines are configured from a section ofcapillary tubing whose length and diameter are optimized to provide thenecessary volume within the capillary tubing to minimize flow rateperturbations.

[0052] Although the chromatography pumping system described in theillustrative embodiment herein is configured to accommodate two separatesolvent sources it should be appreciated that multiple or single solventdelivery systems as are known in the art can be implemented.

[0053] Although the chromatography pumping system described in theillustrative embodiment herein is configured having traditional actuatorand spring backflow preventors it should be appreciated that otherbackflow preventors that are known in the art can be used.

[0054] The foregoing describes specific embodiments of the inventivemethod and apparatus. The present disclosure is not limited in scope bythe illustrative embodiments described, which are intended as specificillustrations of individual aspects of the disclosure. Equivalentmethods and components are within the scope of the disclosure. Indeed,the instant disclosure permits various and further modifications to theillustrative embodiments, which will become apparent to those skilled inthe art. Such modifications are intended to fall within the scope of theappended claims.

What is claimed is:
 1. A method for improving the compositional accuracyof high pressure gradient pumps for high pressure liquid chromatographycomprising the steps of: providing a first solvent line having a firstset of pumps and a second solvent line having a second set of pumps saidfirst set of pumps being in fluid communication with to a first solventreservoir and said second set of pumps being in fluid communication witha second solvent reservoir; connecting said first set of pumps to afirst pulse dampener and connecting said second set of pumps to a secondpulse dampener said first pulse dampener being in fluid communicationwith a first backflow preventor and said second pulse dampener being influid communication with a second blackflow preventor wherein saidbackflow preventors substantially reduce fluidic cross talk between saidsolvent lines; connecting said backflow preventors to a mixing tee todeliver solvent composition.
 2. The method of backflow preventionaccording to claim 1 wherein said backflow preventors improvecompositional accuracy.
 3. The method of backflow regulation accordingto claim 1 wherein said backflow preventors allow accurate compositionaldelivery at flow rates substantially less than the volumes of said pulsedampeners.
 4. The method of backflow regulation according to claim 1wherein said backflow preventors allow accurate compositional deliveryat flow rates substantially equivalent to displaced volumes of each pumpchamber of said pumps.
 5. The method of backflow prevention according toclaim 1 wherein said backflow preventors are incorporated in said mixingtee to reduce delay volume.
 6. The method of backflow preventionaccording to claim 5 wherein said incorporated backflow preventors havebackpressure regulators.
 7. The method of backflow prevention accordingto claim 1 further comprising the step of providing backpressureregulators having a selected fixed resistance said selected fixedresistance ensuring that said pulse dampeners function efficiently withconsistent performance of primary check valves of said pumps.
 8. A highpressure liquid chromatography apparatus comprising: a first set ofpumps and a second set of pumps said first set of pumps being in fluidcommunication to a first purge valve and said second set of pumps beingin fluid communication to a second purge valve; a first pulse dampenerand a second pulse dampener said first pulse dampener being in fluidcommunication with a first backflow preventor and said second pulsedampener being in fluid communication to second backflow preventor; amixing tee said mixing tee being in fluid communication with saidbackflow preventors and being in fluid communication with a vent valvesaid vent valve being in fluid communication with a chromatographycolumn.
 9. The apparatus for backflow prevention according to claim 8wherein said backflow preventors have backpressure regulators having aselected fixed resistance said selected fixed resistance ensuring thatsaid pulse dampeners function efficiently with consistent performance ofprimary check valves of said pumps.
 10. The apparatus for backflowprevention according to claim 8 wherein said backflow preventors improveaccuracy of compositional delivery.
 11. The apparatus for backflowprevention according to claim 8 wherein said backflow preventors allowaccurate compositional delivery at flow rates substantially equivalentto displaced volumes of each pump chamber of said pumps.
 12. Theapparatus for backflow prevention according to claim 8 wherein saidbackflow regulators are incorporated in said mixing tee to reduce delayvolume.
 13. The apparatus of backflow prevention according to claim 8wherein said incorporated backflow preventors have backpressureregulators.
 14. The apparatus of backflow prevention according to claim8 wherein said backflow preventors allow accurate compositional deliveryat flow rates substantially less than the volumes of said pulsedampeners.
 15. The apparatus for backflow prevention according to claim8 wherein said pulse dampeners reduce pump pulsations thereby reducingvolumetric requirement for effective solvent mixing allowing for the useof high pressure gradient systems in smaller volume chromatographycolumns.
 16. A high pressure liquid chromatography apparatus comprising:a first set of pumps within a first solvent delivery line and a secondset of pumps within a second solvent delivery line said first set ofpumps being in fluid communication with a first solvent reservoir andsaid second set of pumps being in fluid communication with a secondsolvent reservoir; means for pulse dampening so that flow rateperturbations produce by said first set of pumps and said second set ofpumps are reduced; and means for backflow prevention so that fluidiccross talk is eliminated between said solvent delivery lines.
 17. Theapparatus according to claim 16 further comprising means forback-pressure regulation.
 18. A high pressure liquid chromatographyapparatus comprising: a mixing tee having an outlet port, a first inletport and a second inlet port; and a backflow preventor incorporatedwithin said first and second inlet port said backflow preventors havinga selected coil spring and an actuator seated within a valve seatwherein said selected coil spring exerts a selected pressure upon saidactuator.
 19. The high pressure liquid chromatography apparatusaccording to claim 18 wherein said selected pressure is ranges between 0and 2000 psi.
 20. The high pressure liquid chromatography apparatusaccording to claim 18 wherein said incorporation reduces system volume.21. The high pressure liquid chromatography apparatus according to claim18 wherein said backflow preventors have a selected fixed resistancesaid selected fixed resistance ensuring that pulse dampeners within achromatography system function efficiently with consistent performanceof primary check valves of system pumps.
 22. A high pressure liquidchromatography apparatus comprising: a mixing tee having an outlet port,a first inlet port and a second inlet port; and a backflow preventorincorporated within said first and second inlet port said backflowpreventors having a ball bearing seated within a valve seat wherein saidball bearing limits fluid flow to a forward direction.